Symposium 1997: Abstract I for C Roelant et al

Symposium 1997: Abstracts

The Development of a Dual Glow-Signal Firefly and Renilla Luciferase Assay for High Throughput Compound Screening

Chris Roelant¹*, Sue Brown², Jenny Stables², Stephen Rees², Dave Burns³
¹RTRT, Leuven, Belgium,
²Receptor Systems, Glaxo Wellcome Research and Development, Stevenage, UK,
³Packard Instrument Company, Meriden, CT, USA

A dual glow signal firefly and Renilla luciferase microplate assay has been developed. Firefly luciferase activity is first assayed using the LucLite^TM component of this new dual signal kit from Packard. Following this first measurement, the second RenLite^TM component of the kit is added to the same assay samples. This Renilla substrate quenches the firefly luciferase reaction and initiates the Renilla signal to produce a second glow luminescence signal.

We demonstrate the use of this assay for high throughput compound screening for agonists of G-protein coupled receptors which signal through activation of the G-protein sub-unit Gas.

Chinese Hamster Ovary cells expressing either the Adenosine A2b receptor or the beta-2-adrenoceptor were transfected with cAMP responsive reporter constructs containing the firefly and Renilla luciferase's respectively. The two cell lines were mixed in an individual well of a 96-well plate and a range of compounds screened to determine their activity at both receptors.

This assay allows the simultaneous screening of two receptors in a 96-well format resulting in significant cost and time savings. Furthermore the assay could be used to study two signal transduction events within the same cell.